Hematopoietic cell transplantation is potentially curative for a variety of hematologic cancers. However, graft vs host disease (GVHD) and the lack of donors limit their wide application. CD4+CD25+ regulatory T cells (Treg) can suppress aberrant immune responses and regulate peripheral T cell homeostasis. We recently show that Treg suppress GVHD without abrogating the graft vs tumor effect in a murine model. However, their rarity and our lack of understanding of their immunoregulation limit their use in the clinical setting. This proposal details a 5-year training program. The long-range goal of this project is to determine whether ex vivo expanded Treg add benefit to HCT. The objective of this project is to evaluate the role of ex vivo expanded Treg in animal models of HCT. The central hypothesis to this work is that Treg can be expanded ex vivo and that such population provide benefits in the post-HCT period. This hypothesis will be tested by pursuing three specific aims: 1) evaluate the in vivo survival and trafficking of ex vivo expanded Treg using ICFSE labeling, flow cytometry, and bioluminescence imaging of Treg from luciferase transgenic mice; 2) evaluate the effects of altered Treg homing and migration on GVHD and the graft vs tumor (GVT) using knockouts of L-selectin and alpha4-beta7 integrin; 3) determine how ex vivo expanded Treg alter the immune reconstitution of the donor graft with spectratype analysis, phenotyping of lymphoid organ and peripheral blood mononuclear cells via flow cytometry, and functional analysis by CMV infection. The proposed work is innovative because we study a rare population of cells using noninvasive techniques in sensitive animal models of GVHD and GVT. It is expected that our studies will provide preclinical information that is important for human studies. The proposed training program is in a supportive and dynamic research setting. Thus, the candidate will gain the necessary skills to become a successful independent investigator in the field of transplant immunology.